1. Field of the Invention
This invention pertains to the expression of proteins which require post-translational modification of their amino acid sequence before a mature form is reached. Such proteins exhibit amino acids other than the 20 common amino acids coded for by the conventional nucleic acids. Specifically, a leader peptide sequence is identified which can induce post-translational modification of specific amino acids when expressed in conjunction with the precursor polypeptide. Methods of forming improved compositions using this leader sequence are also addressed.
This invention also pertains to a method of converting nonbacteriocin expressing Bacillus strains to bacteriocin expressing Bacillus strains, and to mutant forms of subtilin produced by that method. Specifically, a form of subtilin having enhanced stability and activity is addressed, together with the gene therefor, and expression vehicles for that gene.
2. Discussion of the Background
Polypeptides, including those having natural antibiotic activities, have been identified which comprise amino acids other than the 20 common acids specified by the genetic code, as the expression products of bacteria, and other organisms. The structure of two of the more important ones, nisin and subtilin, are set forth in FIG. 1 of this application.
The presence in these polypeptides, and others, of the unusual amino acids lanthionine, .beta.-methyllanthionine, D-alanine, dehydroalanine and dehydrobutyrine clearly suggests that something other than ordinary protein biosynthesis directed by the genetic code is involved in the expression of the mature forms of these naturally occurring polypeptides. Nonetheless, research has demonstrated that the appearance of these polypeptides can be blocked by protein biosynthesis inhibitors. Hurst et al., Canadian Journal of Microbiology, 17, 1379-1384 (1971). It is also known that precursor peptides of the mature forms can be detected with antibodies against the mature peptide. Nishio et al., Biochemistry Biophysics Research Community, 116, 751 (1983). These observations, with other observations concerning nisin, subtilin and related proteins suggest a mechanism that involves primary biosynthesis of a precursor via a ribosomal mechanism, followed by post-translational modifications.
The activity of these proteins, and potential mutant variations thereof, are of sufficient commercial interest so as to generate substantial activity in the field of derived microorganisms containing foreign DNA fragments and coding for the protein's production. U.S. Pat. No. 4,716,115, issued to Gonzalez et al. is directed to just such a derived microorganism. However, the impossibility of obtaining a genetic sequence that codes directly for the mature protein, and the lack of information concerning the nature of the post-translational modification necessary to arrive at the mature protein, has prohibited the cloning of microorganisms containing the specific gene which encodes for these proteins, and perhaps more importantly, has frustrated attempts to produce random variants and site-specific mutated proteins, which quite probably can be arrived at having higher degrees of activity, or other enhanced properties.
Thus, it remains an object of the biotechnology field to arrive at a comprehensive understanding of the mechanism by which the mature forms of these unusual amino acid-containing polypeptides are made, and to develop an expression vehicle for incorporating a gene which will specifically encode for the production of these peptides and which is suitable for the transformation of commonly available bacteria.
In application Ser. No. 07/214,959 (now U.S. Pat. No. 5,218,101), the polypeptide precursors for expression of mature subtilin and nisin, and corresponding gene sequences, are disclosed. As related in this application, these bacteriocins are of particular interest in that they contain unusual amino acids that are introduced subsequent to nucleic acid translation, presumably by specific enzyme mechanisms contained within the cell, and possibly on the ribosome. Thus, this application identifies the gene and amino acid leader sequence necessary for the expression of the polypeptide precursor which, upon undergoing post-translational modification, results in the expression of the mature bacteriocin.
While these two antibiotics share considerable structural homology, as discussed in application Ser. No. 07/214,959, now U.S. Pat. No. 5,218,101, they are quite distinct in certain chemical properties. Of particular importance is the tendency of the subtilins to undergo spontaneous inactivation at a substantially greater rate than that exhibited by nisin. In aqueous solution at pH 6.8, spontaneous inactivation is accompanied by chemical modification of the dehydroalanine at position 5 of the mature bacteriocin, with a kinetic first-order t.sub.1/2 of 0.8 days. It is noted that the amino acid in the four position, Glu, bares an R-group on its carboxyl moiety, which may participate in the chemical modification of the adjacent amino acid residue.
Thus, nisin, which is resistant to inactivation at low pH and high temperatures, Hurst, Advanced Application of Microbiology, volume 27, pages 85-123 (1981) is widely used as a food preservative, Hurst, supra as well as Jay, Food Microbiology, vol. 8, pages 117-143 (1983) and a treatment for bacterial infections, Sears et al, Journal of Diary Science 74, page 203 (1991). In contrast, subtilin's instability renders it of little practical value, despite having a broad spectrum of action. Jay, supra.
It is clearly a desire of those of skill in the art to obtain a mature form of subtilin which is resistant to inactivation and exhibits reasonable activity, to provide an antibiotic with the potential utility of nisin. This is particularly important in light of the increasing antibiotic resistance observed among microbial populations due to the widespread use of existing antibiotics. It is further desirable of producing subtilin forms from a Bacillus host to obtain improvements in yield, and take advantage of developments.